We propose that existing beta-lactam scaffolds can be modified to address the emerging threat of Gram- negative resistance. A wealth of beta-lactams were synthesized from the 1950s into the 1980s but were never developed into antimicrobial drugs. Under the current stress of increasing drug resistance, and with decades of accrued medicinal chemistry knowledge at our fingertips, certain of these molecules are of renewed interest as a result of their non-susceptibility to resistance mechanisms that have since evolved. The utility of beta-lactam antibiotics has been compromised by the spread of beta-lactamase enzymes, both of the serine type (Classes A, C and D) and the metallo type (Class B). The carbapenems, while stable to most Class A and Class C beta-lactamases, are susceptible to Class B enzymes as well as emerging Klebsiella pneumoniae carbapenemase (KPC) Class A enzymes. Monobactams, by contrast, are intrinsically stable to Class B metalloenzymes, and we have synthesized a prototype compound that is additionally stable to KPC enzymes. A new agent that is not susceptible to any of these enzymes would have a major impact on restoring the utility of the beta-lactam class. We have evaluated numerous existing, older beta-lactam agents against panels of bacteria that are well-characterized with respect to expression of beta-lactamase enzymes as well as efflux pumps. The Structure-Activity Relationships (SAR) from this data set have enabled us to design several series of new beta-lactam molecules that are expected to retain potent activity against organisms that are resistant to currently-available agents. We hypothesize that we can modify a carefully selected scaffold that is active against pathogens producing KPC enzymes to include moieties that would make it potent against Class A and Class C beta-lactamases. To accomplish this, we propose two aims in the exploratory R21 phase of the project to provide us with our drug candidate. Then we propose three development aims in the R33 phase. The aims for the R21 phase include: Aim 1. Optimize the in vitro activity of the selected lead compounds with respect to spectrum and potency. Aim 2. Conduct pharmacokinetics (PK) studies and optimize efficacy in infection models. Aims to be conducted during R33 phase include: Aim 3. Conduct higher order pharmacokinetic studies. Aim 4. Conduct in vitro toxicology battery and pilot toxicology studies. Aim 5. Conduct 14-day range-finding toxicology studies. Upon successful completion of this project, we will be ready to proceed into IND-enabling toxicology studies. PUBLIC HEALTH RELEVANCE: Monobactams are penicillin-like drugs that were used to treat bacterial illness in the past. Though their development lagged with the advent of new classes of antibiotics, emerging drug-resistance to such newer classes has renewed our interest in monobactams. We propose to take an older monobactam scaffold and modify it using more recently developed chemistry to create a novel drug that can address difficult, drug-resistant bacterial infections.